This invention relates to a process of entrapping genetic materials in nanoparticles of inorganic compounds of size below 100 nm diameter to form non-viral carriers suitable for delivery of genes including those of therapeutic interest in appropriate cells.
As it is known, the ability to safely and efficiently transfer foreign DNA into cells is a fundamental goal in biotechnology. In recent years, with the advent of recombinant DNA technology, a surge in research activity has occurred in the field of DNA transfer across cell lines. This activity, which has taken the shape of what is popularly known as gene therapy, is a medical/surgical intervention technique which is being developed as a xe2x80x98molecular medicinexe2x80x99 and requires genes to be introduced into cells in order to treat a wide variety of till now incurable human diseases. Potential applications are numerous, given the diversity of the genes to be used as well as the possible target cells.
Today""s gene therapy research may be seen as pursuing intelligent drug design through a logical extension of results of fundamental biomedical research on the molecular basis of disease. The term gene therapy applies to approaches to disease treatment based on the insertion of genetic material (DNA and RNA) into a cell""s genetic pool either to correct an underlying defect or to modify the characteristics of a cell via expression of the newly inserted gene. In order to successfully implement this technique, effective means of delivering the therapeutic gene to the target cell is required, in such a way that the gene can be expressed at the appropriate level and for a sufficient duration. Two broad approaches have been used to deliver DNA and RNA to cells, namely viral and non-viral vectors, which have different advantages as regards efficiency, ease of production and safety. One of the most powerful methods for gene-transfer is the use of viral vectors. A viral vector is genetically engineered from xe2x80x98wild-typexe2x80x99 virus, and consists of a modified viral genome and virion structure. By retaining the protein coat of the original virus, the vector is able to bind and penetrate the cell more effectively while protecting the genome from endogenous enzymes. As for the original viral genome (wild-type), only the essential viral sequence necessary for transcription is retained. There are a number of viral vectors that are currently being used for transfecting cells. Of interest are retroviruses (enveloped single strand RNA), adenoviruses (non-enveloped double stranded DNA) and adeno-associated viruses (linear single stranded DNA). Due to their inherent nature of penetrating and inserting their genetic material (genome) into the target cell, viral vectors result in very high transfection rates. In addition to escaping the target cell""s endonucleases, viral genes also possess promoters and enhancers that increase the probability of genetic expression.
Although viral vectors are attractive in terms of the scientific strategy of exploiting natural mechanism, there are some major drawbacks associated with them. They suffer from inherent difficulties of effective pharmaceutical processing, immunogenicity, difficulty in targeting to specific cell types, scale up and the possibility of reversion of an engineered virus to the wild type The safety risks include xe2x80x98Insertional Mutagenesisxe2x80x99 and toxicity problems. Ever since the death of Jesse Gelsinger in September 2000, scientists have began to severely question the safety aspects related to viral vector mediated gene delivery. Consequently, a major focus is now being given at the development and use of alternative vectors based on synthetic, non-viral systems for safe and efficient gene delivery.
The problems associated with viral vectors have led to a growing interest in non-viral gene delivery systems. Non-viral vectors are techniques of introducing a coding DNA sequence without the means of a virus. The self-assembly of artificial plasmid (pDNA) containing vectors is required for the development of such vectors. These methods of gene transfer require only a small number of gene, have a virtually infinite capacity, have no infectious or mutagenic capability and large scale production is possible using pharmaceutical techniques. DNA itself is negatively charged, as is the cell membrane and therefore the entry of naked DNA is restricted due to electrical repulsion forces. To reduce this repulsion, many researchers have encased the polynucleotide with a cationic membrane so as to alter the electrical distribution and charge of the complex. These include lipid-based carriers, polycationic lipids, polylysine, polyornithine, histones and other chromosomal proteins, hydrogel polymers and precipitated calcium phosphate (CaPi). One of the major drawbacks of the use of these non-viral vectors is their low transfection efficiency which is caused due to exposure of DNA in the hostile DNAse environment due to simple electrostatic compaction of DNA with the polymeric materials. Among these, the technique of calcium phosphate co-precipitation for in vitro transfection is used as a routine laboratory procedure. This procedure involves a reaction of calcium chloride with sodium phosphate to form a water insoluble calcium phosphate precipitate, which can bind to pDNA. This method heavily relies on the fact that divalent metal cations, such as Ca2+, Mg2+, Mn2+and Ba2+can form ionic complexes with the helical phosphates of DNA. Calcium phosphate, therefore, forms complexes with the nucleic acid backbone and thus may impart a stabilizing function to certain DNA structures. When added to a cell monolayer, the cells take up the water insoluble calcium phosphate-pDNA complex (Ca Pi-pDNA) by transportation across the membrane through Ca2+ion mediated channel formation. This process is an example of ion channel mediated endocytosis. Once inside the cell, the CaPi-pDNA complex is broken down inside the endosome, thereby releasing the pDNA into the cytosol, which, under suitable circumstances, can be incorporated into the host cell genome. In addition, being inorganic particles, calcium phosphate is highly stable, non-toxic, non-antigenic and non-carcinogenic.
Although extremely safe, the major shortcoming of this process is the poor transfection efficiency as compared to that of viral vectors. The general belief is that the transfection with CaPi-DNA is a low efficiency procedure partly because most of the endocytosed DNA is quickly degraded and excreted to the cytosol. A small fraction of the remaining DNA macromolecules important for gene transfer may be delivered from the endosomal compartment through membrane bound organelles to the nucleus without traversing the cytosol. Moreover, although calcium phosphate precipitation method is simple, effective and still widely used in laboratory for in vitro transfection, the method is hampered by the difficulty of applying to in vivo studies, especially delivery of DNA to any particular cell types. Due to bulk precipitation of calcium phosphate, the method also suffers from variation in calcium phosphate-DNA particle size, which causes variation among experiments.
Process for production of inorganic nanoparticles has been described in U.S. Pat. Nos. 5,460,831 and 5,879,715. Although the process has described the method of preparation of particles of size as small as 10 nm diameter the preparative method does not describe anything about the encapsulation of biologically active materials inside the matrices of these nanoparticles. Calcium Phosphate nanoparticles of size 300 nm and above have been reported in U.S. Pat. No. 6,355,271, which have been, used as carriers and as controlled release matrices for biologically active materials. Virus-like-size particles i.e. particles of size below 100 nm diameter encapsulating genetic materials, which are biologically safe and cost effective, are the main criteria of a non-viral vector for effective delivery of genes. We have described in this invention of the preparation of below 100 nm diameter inorganic nanoparticles doped with genetic material such as DNA or RNA as a non-viral carrier for the delivery of genes or their modified compounds.
The object of this invention is to propose a novel process for the preparation of nearly monodispersed non-toxic and biocompatible inorganic materials such as calcium, magnesium, manganous phosphates and the like, encapsulating genetic materials such as DNA and RNA and having a size maximum upto 100 nm diameter with near monodispersity.
Another object of this invention is to propose a process for the preparation of nearly monodispersed inorganic nanoparticles of subcolloidal size with targeted DNA and RNA materials. Yet another object of this invention is to propose a process for the preparation of nearly monodispersed inorganic nanoparticles dispersed in aqueous buffer and free from any toxic materials.
Further object of this invention is to propose a process for the complete encapsulation of the therapeutic genetic material into the matrix of the inorganic nanoparticles to secure them from outer intervention in vivo or cell culture in vitro till they are exposed to the target site within the cell.
A still further object of this invention is to propose a process for the preparation of nearly monodispersed DNA or RNA loaded inorganic nanoparticles covered with strongly adhesive and non-toxic biocompatible polymeric material chemically conjugated with targetable ligand so that the particles can be targeted to specific cell types in vivo, which obviates the disadvantages associated with these of the prior art.
To achieve these objectives, this invention provides a process of entrapping genetic materials in nanoparticles of inorganic compounds of size below 100 nm diameter to form non-viral carriers suitable for delivery of genes including those of therapeutic interest comprising the steps of:
(a) dissolving 0.01M to 1.0M of a surfactant or a mixture of surfactant and a cosurfactant in oil to obtain reverse micelles,
(b) adding an aqueous solution of genetic material to the reverse micelles,
(c) dividing the reverse micelles obtained in step (b) into two equal parts,
(d) dissolving aqueous solution of 0.1 to 1.0M inorganic metal salts in one part of reverse micelles (step c) to obtain optically clear and transparent reverse micelles after dissolution,
(e) adding aqueous solution of 0.1 to 1.0M precipitating agent in the second part of reverse micelles (step c) to obtain optically clear and transparent reverse micelles after dissolution,
(f) maintaining the same molar ratio of water to surfactant in steps d and e,
(g) mixing the reverse micelles of both steps (d) and (e) and stirring to form inorganic nanoparticles encapsulating added genetic material,
(h) separating the nanoparticles from reverse micelles, and
(i) dispersing the inorganic nanoparticles in water and dialyzing to remove free metal salts, surfactant and oil.
The above process further comprises coating the nanoparticles surface by adhesive polymeric compound and chemically conjugating ligand molecules for targeting the nanoparticels to specific cell type.
The surfactant is selected from the group containing anionic, cationic and non-ionic type.
The oil used for the preparation of reverse micelles is hydrocarbon oil.
The hydrocarbon oil is a saturated long chain or branched chain hydrocarbon of C6 to C10 chain length.
The hydrocarbon oil is n-Hexane.
The reverse micelles contain a long chain alcohol from butanol to octanol in the form of cosurfactant when it is required to stabilize the reverse micelles.
The genetic materials are selected from DNA and RNA and genetic modifications thereof.
The inorganic metal salts are selected from the group containing calcium chloride, magnesium sulphate and manganous sulphate and the precipitating agent is disodium hydrogen phosphate.
The inorganic metal salt is ferric chloride and the precipitating agent is ammonium hydroxide. The separation of nanoparticle is carried out by precipitating with ethanol and ishing the precipitate with ethanol.
The inorganic nanoparticles are calcium phosphate, magnesium phosphate, manganous phosphate and ferric oxide.
The nanoparticles after separating from micelles be dispersed in water either by mild agitating, prolonged stirring or by sonication.
The molar ratio of water to surfactant (W0) is in the range of W0=10 to W0=40.
The nanaoparticles encapsulating genetic material have diameter in the range of 10 nm to 100 nm.
The adhesive polymeric materials used for coating the nanoparticles is polyacrylic acid.
The ligand is a molecule having at least one amino group selected from the group containing sugar, an antibody, folic acid, transferrin and biotine or derivatives thereof
The carboxylic group of polyacrylic acid is conjugated with the amino group of the ligand molecule.
In accordance with this invention the aqueous core of a reverse micellar droplet is used as a nanoreactor for the preparation of nanoparticles. Near monodispersity of the inorganic particles is possible because reverse micellar droplets in which the precipitation reactions are carried out are highly monodispersed. The size of the nanoparticles is governed by the size of the aqueous core of reverse micellar droplets, which is dependent on the molar ratio of water to surfactant (wo) of the reverse micelles. The wo of reverse micelles is kept in the range of 10 to 40 and the nanoparticles formed in such reverse micelles have average size below 100 nm diameter.
The composition of aqueous phase of reverse micellar droplets is regulated in such a manner so as to keep the entire mixture in an optically transparent reverse micellar phase. The range of aqueous phase can not be defined apriori as this would depend on factors such as nature and solubility of the metal salt used the nature and solubility of the precipitating agent and their interaction with the polar head group of the surfactant. The only factor that is important is that the system should be in an optically transparent reverse micellar phase.
In accordance with the present invention the nanoparticles have size range of upto 100 nm diameter. In accordance with this invention the aqueous core of a reverse micellar droplet having a predetermined wo value is effectively used as nanoreactor to prepare ultrafine nanoparticles and to encapsulate the plasmid DNA, RNA or their derivatives. The process of the present invention has achieved extremely small size nanoparticles (diameter in the range of 10 nm to 100 nm) of greater uniformity.
The strategy involves precipitation of insoluble inorganic metal salts in the form of nanoparticles encapsulating DNA and RNA in the aqueous core of the reverse micellar droplets. As the aqueous core of reverse micelles are of nanosized dimensions, the particles prepared inside them are also nanometer sized. In addition, the aqueous core of reverse micelles has long been known as a medium for solubilizing biomolecules like enzymes, antibodies, other proteins, nucleic acids etc., without damaging their biological activities. In this work, we have achieved inorganic materials to undergo precipitation reaction inside the aqueous core of reverse micelles and have obtained nearly monodispersed nanoparticles completely encapsulating (more than 99%) genetic materials. We have also demonstrated that these nanoparticles doped with genetic material can be used as efficient non-viral vectors for delivery of genes in vitro and in vivo. Because of the extremely low size of the particles, their aqueous dispersion will have easy circulation in the blood. Additionally, these nanoparticles could also be targeted using ligands to receptors of specific cell types in vivo. For this we could cover the surface of these inorganic nanoparticles by some adhesive polymer having a suitable functional group, which can be chemically conjugated with appropriate ligand molecules. Through our invention we have overcome the two major impediments for using inorganic materials as non-viral vectors: (i) use of ultrafine nanoparticles so that the aqueous dispersion of these inorganic salts can become easily injectable systems and (ii) targeting these nanoparticles to specific cell types by coating the nanoparticle surface with adhesive polymer and conjugating them with appropriate ligand. By this way, inorganic nanoparticles mediated gene delivery can become more advantageous compared to other viral and non-viral carriers in the sense that the method is absolutely safe as well as cost effective. With the prospect of reviving this methodology of using calcium phosphate as carriers by improving on the preparative conditions, we have tried to devise a strategy that would make the process more efficient and useful, as well as suitable for in vivo applications.